Regulation and role of the Arabidopsis abscisic acid-insensitive 5 gene in abscisic acid, sugar, and stress response

Abstract

Abscisic acid (ABA) and stress response from late embryonic growth through early seedling development is regulated by a signaling network that includes the Arabidopsis ABA-insensitive (ABI)5 gene, which encodes a basic leucine zipper transcription factor. We have characterized genetic, developmental, and environmental regulation of ABI5 expression. Although expressed most strongly in seeds, the ABI5 promoter is also active in vegetative and floral tissue. Vegetative expression is strongly induced by ABA, and weakly by stress treatments during a limited developmental window up to approximately 2 d post-stratification, but ABA and some stresses can induce expression in specific tissues at later stages. ABI5 expression is autoregulated in transgenic plants and yeast (Saccharomyces cerevisiae), and stress response appears to involve ABI5-dependent and -independent mechanisms. To determine whether ABI5 is necessary and/or sufficient for ABA or stress response, we assayed the effects of increased ABI5 expression on growth and gene expression. Although overexpression of ABI5 confers hypersensitivity to ABA and sugar, as previously described for ABI4 and ABI3 overexpression lines, it has relatively limited effects on enhancing ABA-responsive gene expression. Comparison of expression of eight ABI5-homologous genes shows overlapping regulation by ABI3, ABI4, and ABI5, suggestive of a combinatorial network involving positive and negative regulatory interactions.

Figure 1

ABI5- and developmental regulation of ABI5::GUS activity during early seedling growth. GUS activity was measured in dry seeds or germinating seedlings at 1.5, 3, 6, and 10 d post-stratification. Extracts were derived from isogenic ABI5::GUS transgenic lines, differing only at the ABI5 locus. Activity (nanomoles MU produced per hour) is expressed per plant and per milligram of fresh weight. Values shown are the mean ± sd of eight replicate assays.

Figure 2

ABI5 promoter regulation in yeast. One-hybrid analysis with ABI5 promoter fragments fused to lacZ reporter. A, Promoter fragments used in fusions. Black boxes represent DPBF core recognition sequences. Arrow represents the transcription unit. B, ABI5 promoter-driven β-galactosidase activity produced by GAL4AD fusions to ABI transcription factors (AD-ABIx) or vector control (AD).

Figure 3

Localization of ABI5::GUS activity during development or stress response of wild-type plants. A, Seed with heart-stage embryo; B, cotyledon stage embryo; C, mid-maturation stage embryo; D, mature seed; E, 10-d seedling; F, silique; G, leaf from 3-week-old plant; H, flowers; and I, 3-d-old seedlings treated as indicated: t = 0, or 2 d of exposure to minimal medium (Min); ABA, 50 μm ABA; Glc, 250 mm Glc; Cold, 4°C; NaCl, 125 mm NaCl.

Figure 4

Stress induction of ABI5 promoter activity in seedlings. GUS activity was measured in seedlings at 1.5, 3, 6, and 10 d post-stratification that were harvested immediately (t = 0), dried on absorbent paper in a closed petri dish for 2 h prior to harvest (Dry), or incubated on control medium (min) or exposed to 50 μm ABA, 125 mm NaCl, 250 mm sorbitol, 250 mm Glc, or 4°C for 2 d prior to harvest. Extracts were derived from isogenic ABI5::GUS transgenic lines, differing only at the ABI5 locus. Activity (nanomoles MU per hour) is expressed per milligram of fresh weight. Values shown are the mean ± sd of duplicate assays on two to four samples per treatment.

Figure 5

ABA-responsive gene expression in ABI overexpression lines. Plants were grown for 12 d on GM, then transferred to fresh media ± 50 μm ABA for 2 d prior to harvest. RNA gel blots were hybridized to probes corresponding to the indicated genes. A, ABI5 expression in four independent 35S::ABI5 lines and their corresponding wild-type progenitors. Each lane contains 1 μg of total RNA. B, ABA-inducible marker gene expression in 35S::ABI5 lines, representative strong ABI3 and ABI4 overexpression lines, and all corresponding wild types. Each lane contains 5 μg of total RNA. C, ABI3 and ABI4 expression in 35S::ABI5 and wild-type lines. For ABI3 hybridization, 35S::ABI3 lanes contain 0.5 μg and others contain 10 μg of total RNA. For ABI4 hybridization, 35S::ABI4 lanes contain 1 μg and others contain 15 μg of total RNA.

Figure 6

Effects of ABI transcription factor function on ABA hypersensitivity of root growth. After 2 d of growth on hormone-free medium, seedlings were transferred to fresh media containing 0 or 3 μm ABA. New growth was measured after 4 d and is expressed as a percentage of growth on the control medium. Each set represents the average of at least 10 replicates within an individual experiment; sds are expressed as percentages of the control growth. An asterisk indicates 35S::ABI5 lines with significantly increased ABA sensitivity (P = 0.034, 0.0005, or 4 × 10⁻⁵ for lines 2A2, 2D1, and 2A4, respectively, based on Student's t test).

Figure 7

ABI5 overexpression increases seedling sensitivity to sugar. Seedlings of the indicated genotypes were harvested after 9 d of growth on minimal medium, with or without 4% (w/v) Glc. A, Fresh weight per plant. B, Anthocyanin content per fresh weight. Values shown are the mean ± sd of two to four assays per treatment.

Figure 8

ABI5-homologous bZIP expression in ABI overexpression lines. A, RNA gel-blot analyses of three strong 35S::ABI lines (isolates C7A19, 114A, and 2D1 for ABI3, ABI4, and ABI5 overexpression, respectively) hybridized to cDNAs or PCR fragments corresponding to the indicated genes. All reported names are shown for genes isolated independently in multiple labs. Each lane contains 7 μg of total RNA. B, Tree depicting homology relationships among Arabidopsis ABI5-homologous family members. The corresponding AGI or GenBank accession numbers for the named genes, followed by the bZIP designations proposed by Jakoby et al. (2002), are given in parentheses: AtDPBF2 (At3g44460; AtbZIP67); AtDPBF3/AREB3 (At3g56850; AtbZIP66); AtDPBF4 (At2g41070; AtbZIP12); AtDPBF5/ABF3 (At4g34000; AtbZIP37); ABF1 (At1g49720; AtbZIP35); ABF2/AREB1 (AF093545; AtbZIP36); and ABF4/AREB2 (At3g19290; AtbZIP38). Although ABF2 and AREB1 have been independently cloned as cDNAs, the corresponding genomic sequence has not yet been identified.